1. Ablation of miR-146b in mice causes hematopoietic malignancy
by Takahiro Mitsumura, Yoshiaki Ito, Tomoki Chiba, Takahide Matsushima, Ryota Kurimoto, Yoko Tanaka, Tomomi Kato, Keisuke Uchida, Takashi Ito, Kouhei Yamamoto, Yoshinobu Eishi, Masanobu Kitagawa, Yasunari Miyazaki, Naohiko Inase, and Hiroshi Asahara
BloodAdv
Volume 2(23):
December 11, 2018
© 2018 by The American Society of Hematology

2. Takahiro Mitsumura et al. Blood Adv 2018;2:3483-3491
© 2018 by The American Society of Hematology

3. Production of miR-146a and miR-146b KO mice with TALEN
Production of miR-146a and miR-146b KO mice with TALEN. (A) Genetic deletion in miR-146a– and miR-146b–deficient mice generated by TALEN. The genomic sequence is shown at the top, with the target sequences of a pair of TALEN colored in red.
Production of miR-146a and miR-146b KO mice with TALEN. (A) Genetic deletion in miR-146a– and miR-146b–deficient mice generated by TALEN. The genomic sequence is shown at the top, with the target sequences of a pair of TALEN colored in red. Blue lines and boxes indicate pre-miRNA and mature miRNA sequences, respectively. Deleted nucleotides are indicated with dashes. The sizes of the deletions and insertions are shown to the right of the mutated alleles with Δ. WT indicates alleles without a mutation. (B) RT-PCR analysis of miR-146a and miR-146b in liver tissue derived from mutant mice. The genotype of each animal is indicated at the bottom. The expression level of miR-146a and miR-146b in the WT animal was adjusted to 1.
Takahiro Mitsumura et al. Blood Adv 2018;2:
© 2018 by The American Society of Hematology

4. Histological analysis of spleens and lymph nodes in WT mice and representative malignancies in miR-146a or miR-146b KO mice.
Histological analysis of spleens and lymph nodes in WT mice and representative malignancies in miR-146a or miR-146b KO mice. (A) Increased rate of tumorigenesis in mice with miR-146a or miR-146b ablation. n = 11 (WT), 8 (miR-146a KO1), 3 (miR-146a KO2), and 21 (miR-146b KO). (B) Photograph of spleens isolated from WT and miR-146b KO mice at 16 months. (C) miR-146a and miR-146b KO mice developed B-cell lymphomas in the spleen and lymph nodes. Scale bars represent 500 µm (low magnification), 20 µm (high magnification), and 2.5 µm (insets). (D) miR-146b KO mice developed acute leukemia. Scale bars represent 500 µm (low magnification) and 20 µm (high magnification), and 2.5 µm (inset). HE, hematoxylin and eosin; FL, follicular lymphoma.
Takahiro Mitsumura et al. Blood Adv 2018;2:
© 2018 by The American Society of Hematology

5. miR-146a and miR-146b induction in response to LPS, anti-CD40 antibody, and anti-IgM antibody treatment in splenic B cells.
miR-146a and miR-146b induction in response to LPS, anti-CD40 antibody, and anti-IgM antibody treatment in splenic B cells. Quantitative RT-PCR analysis of mature miR-146a and miR-146b expression in WT splenic B cells in response to 10 μg/mL anti-IgM antibody (A), 1 μg/mL LPS (B), and 0.1 μg/mL anti-CD40 antibody (C). Mature miR-146a and miR-146b expression was normalized to that of snoRNA202. Data are presented as mean ± standard error of the mean (SEM); n = 3. *P < .05, **P < .01, ***P < .001, ****P <
Takahiro Mitsumura et al. Blood Adv 2018;2:
© 2018 by The American Society of Hematology

6. Suppression of NF-κB activity by miR-146a and miR-146b.
Suppression of NF-κB activity by miR-146a and miR-146b. (A) Luciferase reporter assay for miR-146a– or miR-146b–dependent regulation of NF-κB. Luciferase activity was measured in HEK293T cells transfected with a control empty vector, miR-146a, or miR-146b expression vector (with or without 50 ng/mL PMA). Data are presented as the mean ± SEM. Two independent experiments were performed, and similar results were obtained. +, 5 ng; ++, 75 ng. (B) Luciferase reporter assays in 293FT cells transfected with pcDNA-miR-146a (miR-146a), pcDNA-miR-146b (miR-146b), or an empty vector (Ctrl). MCS, pLuc2-KAP-MCS (multicloning site as an empty reporter). Data are presented as the mean ± SEM; n = 3. (C) Western blot analysis of TRAF6 protein expression in nonstimulated WT and miR-146a or miR-146b KO splenic B cells and those stimulated with LPS or anti-CD40 antibody. Two mice per genotype were analyzed; representative results are shown. *P < .05, **P < .01.
Takahiro Mitsumura et al. Blood Adv 2018;2:
© 2018 by The American Society of Hematology

7. Proliferation of WT, miR-146a KO, and miR-146b KO B cells in response to LPS, anti-CD40, and anti-IgM antibody.
Proliferation of WT, miR-146a KO, and miR-146b KO B cells in response to LPS, anti-CD40, and anti-IgM antibody. Splenic B cells (CD19+) were incubated for 48 hours in the presence of media only (none) or the indicated mitogens (0.1, 1, or 10 μg/mL LPS; 0.1, 1, or 5 μg/mL anti-CD40; and 10 μg/mL anti-IgM). Proliferation was measured by [3H]-thymidine incorporation. Data are presented as mean ± SEM. n = 6. *P < .05, **P < .01. cpm, counts per minute.
Takahiro Mitsumura et al. Blood Adv 2018;2:
© 2018 by The American Society of Hematology